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- Posts: 4
- Joined: Thu Jan 31, 2019 5:39 pm
I am looking at acetic, propanoic, butyric, isobutyric, valeric, isovaleric, and using heptanoic acid as a surrogate. Naphthalene is being used for the internal standard.
I am evaluating two separate derivation methods as part of the project, BSTFA and PFBBr. So far I've diluted the SCFA's in diethyl ether and derivatized them. I'm getting good separation, and now it's time to build a calibration curve.
The PI suggested spiking the SCFA's into serum to build the calibration curve (he's a microbiologist, not a chemist—but then, I'm just an undergrad, so...), but since I'm extracting out into diethyl ether, and my evaluation is of the organic layer, that doesn't make sense to me, since there might be variable loss in the process, and then, what are you really calibrating?
My thought is that it should be derivatized SCFAs in diethyl ether, but I believe Peter Apps mentioned in one of the posts that analytes in solvent weren't a good starting point for calibration. Anyone care to elucidate?
My second question is, I suspect I want to make my dilutions for calibration from the derivatized sample. The other alternative is to make the dilutions of the SCFAs in solvent, and then to add the derivatizing reagent to each dilution. Which to do?
Thirdly, do I want to treat each SCFA separately for building the calibration curve(s), or do I want to have a single set of dilutions that contains all of the SCFAs in one batch?
And finally, since Naphthalene is being added at the end of the extraction process, do I need to add it to the calibration dilutions?
Cheers,
Chris Gursche